1. Field of the Invention
The present invention relates to an apparatus to address reflection losses associated with microtiter plate readers. More particularly, the present invention relates to modified calibration artifacts for the purpose of calibrating and/or monitoring the calibration of, microtiter plate readers.
2. Description of the Prior Art
Microtiter plate readers are a form of absorbance measuring spectrophotometer, adapted to make their readings vertically through the wells of a microtiter plate containing liquid. Typically, the wells of the microtiter plate are open at the top, so the beam enters (or exits depending on the design) through an air-liquid interface. The bottoms of the wells may be plastic or glass, depending on the design. Thus, as a light beam traverses the microtiter plate, it passes through three interfaces on the way through: air-liquid at the top surface, liquid-solid at the inside bottom of the well and solid-air at the outside of the bottom of the well. This differs from a cuvette in a horizontal beam spectrophotometer which necessarily has four interfaces through which the beam travels. This difference is one of the reasons for discrepancy between readings made in a cuvette using a horizontal beam and a microtiter plate using a vertical beam.
Other reasons for differences in readings between spectrophotometer and plate reader are more related to economy of design and speed of reading. Microtiter plates contain a multiplicity of wells, generally at least 96, so the readers are configured to make their readings quickly to minimize overall read time, sometimes leading to compromises in the optical design and accuracy of results. For one group of readers tested, the results of measurements at A=0.2 in a microtiter plate varied between readers and between channels of readers by up to 8%, a variability far greater than would ordinarily be found in readings of different spectrophotometers. Such differences may be too great to reconcile a true reading and, therefore, exceed an established accuracy specification. In particular, it may negatively impact any comparison made between any two spectrophotometers or any two channels of a multichannel reader.
In an absorbance measuring plate reader, a collimated beam of light is directed (usually downward) onto a plate. A portion passes through the plate and is detected by a detector usually under the plate. Wavelength selection can occur by filter or monochromator and take place either above or below the plate. Some of the light is invariably lost from the beam as it passes through the plate. In addition to the absorbance taking place in the plate or its contents, some of the incident light is lost from the beam because it is reflected at each interface between different optical media. The reflected light will be directed upward back toward the source. In the ideal case, none of the reflected light winds up in the detector. In the case where the plate contents do not absorb, the “absorbance” of the plate as measured by the reader is actually a misnomer, because the light is not absorbed, merely removed from the original beam.
The intensity of light reflected from the boundary between two plane optical surfaces was theoretically derived by Augustin-Jean Fresnel in 1821. The relationship has been experimentally confirmed countless times since then, and holds under a wide range of conditions. In the simplest case, that of a well collimated beam of unpolarized light falling normally (at right angles) to a surface, the amount of light reflected at that surface is given by:
                    R        =                              (                                                            n                  1                                -                                  n                  2                                                                              n                  1                                +                                  n                  2                                                      )                    2                                    (        1.1        )            The quantities ni are the indices of refraction of light on either side of the boundary. The index of refraction is the ratio of the speed of light in the medium compared to that in vacuum. The reflection is the same whether the light is going one way through the boundary or the other. The balance of the beam of light (the part not reflected) is transmitted through the boundary and continues onward. In the case of normal incidence, the transmitted beam is also normal to the surface.
The reflections that naturally occur at all interfaces in a spectrophotometer can result in measurement errors. It is known in the art that multiple reflections of light from cuvette walls can cause errors in absorbance measurement results. Reflections occur from the various optical elements in the instrument and from the sample container. In a high quality spectrophotometer, the optical elements of the instrument are either anti-reflection coated to avoid reflections, or angled to direct reflections away from the detector. This leaves the sample as the principle source of reflection error. The reflections which lead to error are those which pass through the sample three times before reaching the detector. This occurs via the multiple reflection path from the exterior surface of the exit side of the sample, back to the exterior of the input side, and back again through the exit to the detector. In this case, the errors due to reflection will typically amount to about 0.3% at low absorbances, where the largest influence of reflection errors occurs for small absorbance readings (typically <0.5).
In some plate readers, the errors due to reflections are many times greater than this, due to compromises in the optical design to increase the throughput and decrease the cost. For instance, if the detector is: a) close to the microtiter plate, b) made of silicon, as most are, or c) normal to the beam axis, then it will reflect about 30% of the light incident on it back toward the plate. This back reflected light has the opportunity to be multiply reflected and wind up being detected after three passes through the sample. This component of reflected light which makes three passes through the sample before detection is what causes reflection errors in the results. The magnitude of each contribution to the three-pass multiple reflection paths are generally dictated by the types of surfaces present. Thus, the reflections from a cuvette are quite different from the reflections associated with a microtiter plate filled with water, for example, dependent upon the indices of refraction of the different layers described herein.
Attempts have been made to compensate for differences between plate readers. For example, U.S. Pat. No. 7,061,608 describes the use of special cuvettes as artifacts. The entire content of that patent is incorporated herein by reference. However, the special cuvettes may not be sufficient to compensate for reflection loss differences that exist between the cuvette and a sample-filled microtiter plate. These special cuvettes also may not be sufficient to compensate for the different reflection losses experienced among all types of different plate readers provided by multiple manufacturers. Therefore, what is needed is an artifact apparatus to address the difference in reflection losses between a cuvette and a solution-filled microtiter plate. This artifact should be applicable across a plurality of plate reader designs. In particular, what is needed is an artifact apparatus that may be used to calibrate and/or monitor a spectrophotometer by accounting for those reflection losses as a function of the liquid used in calibration solutions.